The central objective of this proposal is to determine mechanisms of entry for human metapneumovirus (hMPV), a recently described paramyxovirus that is a major cause of lower respiratory tract infection in infants and children worldwide. Understanding the virus-cell interactions that lead to docking and internalization of hMPV is critical to elucidating mechanisms of pathogenesis. Host cell receptors are important determinants of virus tropism and can evoke signaling pathways that lead to virus internalization or prime the cell for viral replication. Furthermore, an understanding of virus-receptor interactions may lead to development of targeted antiviral compounds, such as pleconaril, which blocks picornavirus binding to its receptor. Several pathogenic human viruses utilize RGD-binding integrins as receptors or co-receptors, including adenovirus, Coxsackievirus, parechovirus, rotavirus and human herpes virus-8. Our preliminary data suggest that RGD- binding integrins serve as receptors for hMPV and that the fusion (F) protein mediates this interaction. In Aim 1, we will define mechanisms by which integrins mediate hMPV cell entry. hMPV infectivity requires 1V21 integrin, and this interaction is mediated by hMPV F protein. We hypothesize that hMPV F-integrin interaction is not a simple binding event, but that integrin ligation triggers fusion. Wild type and mutant recombinant F protein will be tested for the capacity to bind 1V21 integrin in flow cytometry and surface plasmon resonance assays. Mutant viruses with alterations in integrin-binding motifs will be generated by reverse genetics and tested for the capacity to bind 1V21 integrin and infect cells. The contribution of integrin binding to membrane fusion will be determined using a fusion assay based on a stably transfected cell line expressing wild type or RGD-mutant hMPV F. These experiments will determine the role of 1V21 integrin in hMPV cell attachment, fusion and entry. In Aim 2, we will determine whether integrin engagement by hMPV F triggers intracellular signaling. Integrins mediate both outside-in and inside-out signaling. These experiments will determine whether integrin activation is required for hMPV fusion and cell entry and whether downstream signaling is mediated by hMPV F-integrin binding. We will use experimental conditions that promote or hinder integrin activation to determine whether integrin activation is required for F binding, for fusion and for cell entry. These three steps in hMPV entry will be determined using a recombinant F protein binding flow cytometry assay, a cell-based fusion assay, and hMPV virus infectivity assay. Separate experiments will be performed to determine the effect of F binding on downstream signaling such as focal adhesion kinase (FAK) phosphorylation and the recruitment of second mediators to 1V21 integrins. Inhibitors of downstream integrin signaling, immunoprecipitation and siRNA techniques will be used to determine whether integrin-mediated signaling is required for hMPV entry or replication. These experiments will contribute broadly to an understanding of how integrin signaling properties are utilized by pathogenic viruses. PUBLIC HEALTH RELEVANCE: Human metapneumovirus (hMPV) is an emerging virus that was discovered in 2001. hMPV is a major cause of respiratory tract disease worldwide, and leads to many hospitalizations of infants and children. The purpose of this proposal is to determine the protein (or "receptor") on human cells that hMPV attaches to in order to enter and infect the cell. These studies will increase understanding of how hMPV causes disease in humans, and may help develop targets for antiviral medicines and vaccines.